The aim of this project is to identify and characterize factors that regulate gene expression in vertebrate embryos as an approach to understanding the mechanisms that control early development. The primary model system is the transcriptional regulation of an epidermal keratin gene, XK81A1, in the amphibian (Xenopus laevis) embryo. The cis-regulatory elements of this gene have been mapped by introducing mutated DNA constructs into frog embryos. A positive transcription factor, KTF-1, has been identified which binds to a specific regulatory site and stimulates the activity of the keratin promoter. KTF-1 has been partially purified by affinity chromatography, and its characterization is being continued. Other potential regulatory elements in the keratin promoter are being mapped by various approaches, including PCR-amplified in vivo footprinting. By this latter method several potentially interesting sites of DNA-protein interaction have been identified, including sequences resembling the binding sites for the mammalian transcription factors AP-1 and AP-2. Molecular clones for these factors are available and can be used to, clone Xenopus homologs. In collaboration with Dr. Sheryl Sato (Clinical Endocrinology Branch, NIDDK) the investigation of the posterior-specific putative zinc finger protein called Xpo (Xenopus posterior) is being continued. Xpo can be induced in ectoderm by a combination of mesoderm inducer (such as Activin A or Fibroblast Growth Factor) and retinoic acid. The possible role of Xpo in controlling axis formation is being investigated by injection of synthetic mRNA into fertilized eggs.